The production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.
Mad7 is a CRISPR enzyme, similar to Cas9 and Cas12a, which is of great
interest to industry and academia due to its permissive licensing
agreement, which states that everyone is free to use it but not
distribute it commercially. This paper demonstrates that Mad7 genome
editing works in CHO cells and provides the CHO engineering community
with the computational tools needed to generate Mad7 compatible gRNAs
against the CHO genome.
Mad7 is a CRISPR enzyme, similar to Cas9 and Cas12a, which is of great interest to industry and academia due to its permissive licensing agreement, which states that everyone is free to use it but not distribute it commercially. This paper demonstrates that Mad7 genome editing works in CHO cells and provides the CHO engineering community with the computational tools needed to generate Mad7 compatible gRNAs against the CHO genome.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.